Project Summary/Abstract. Neurons are exceptionally large and exceptionally long-lived. They thus face unusual challenges when it comes to regulating their proteome in time and space. Regulated proteostasis is required to establish, remodel, and maintain the complex dendritic arbors and synapses. In addition, neurons needtoavoidbuild-upofintracellularwasteovertimewhichcanbecometoxic.Notsurprisinglythen,dysfunction oflysosomalpathwayscanleadtonervoussystemdysfunction.Thetraffickingoflysosomalconstituentsiswell studiedinfibroblastswherelysosomalproteases,suchascathepsins,areinitiallydeliveredtoearlyendosomes by both mannose-6-phosphate (M6P)-dependent and ?independent pathways. Subsequent delivery to lysosomes occurs from early and late endosomes. However, significant gaps in our knowledge exist with respect to how the delivery of both cathepsins and lysosome-associated membrane proteins (LAMPs) are spatiallycoordinatedindendrites.Thepremiseforthisapplicationrestsonourownrecentdata: 1)Degradativecapacityalongdendritesishighproximallyandverylowdistally. Recentpublicationsdescribetheimportanceoflysosomestravelingalongdendritesandevenintospines.Inour own work, we discovered that terminal degradation of short-lived dendritic membrane proteins did not occur locally along dendrites. Rather, it overwhelmingly took place in degradative lysosomes clustered in the soma andinthefirst25moftheproximalmajordendrite. 2)Differentconstituentsoflysosomesarefoundindistinctspatialpatterns. In fibroblasts, all cathepsins traffic to lysosomes by the same pathway. Surprisingly, our data show that CatD shows a different distribution from CatB. Furthermore, the popular marker LAMP1, is not a reliable marker of lysosomes in distal dendrites, but found distally in compartments devoid of active cathepsins. There is thus divergence of multiple lysosomal constituents to distinct compartments in the soma and along dendrites, both forLAMP1vsCatB,andforCatBvsCatD. Wewilltestthemechanismsfordivergentpathwaysoflysosomebiogenesisindendritesintwospecificaims: Specific Aim 1: We will test the hypothesis that CatB is delivered directly to soma-near early endosomes via MPRs,butdoesnottraveltodistaldendrites,whereasLAMP1istraffickedthroughoutthelengthsofdendrites toearlyandlateendosomes.TheconvergencepointofLAMP1andCatBtoformdegradativelysosomesmight thusbeasoma-nearlateendosomeratherthanearlyendosomes,asthefibroblastliteraturewouldsuggest. SpecificAim2:WewilltestthehypothesisthatCatD,andLusedistinctmachineryfromCatBtoestablishtheir spatialdendriticprofiles,inparticularMPR-independentpathways. Our long-term goal is to understand the regulation of neuronal proteostasis in space along dendrites and to uncoverthephysiologicalconsequencesofspatiallydiversifiedendo-lysosomesinhealthanddisease.